Preparation method and efficacy evaluation method of swine mycoplasmal pneumonia inactivated vaccine
By optimizing the fermentation process and using efficient crushing technology, a high-titer inactivated Mycoplasma hyopneumoniae vaccine was prepared, solving the problems of harsh culture conditions and unscientific evaluation methods, and achieving efficient and safe vaccine preparation and scientific efficacy evaluation.
Patent Information
- Application Number
- CN202511110365.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-08
- Publication Date
- 2025-11-14
AI Technical Summary
Existing porcine mycoplasma pneumonia vaccines require demanding culture conditions, making efficient preparation difficult. Furthermore, vaccine efficacy evaluation methods are greatly affected by experimental differences and subjective factors, impacting the scientific validity and effectiveness of the vaccines.
By optimizing the fermentation process of Mycoplasma hyopneumoniae, using coenzyme, PPLO, glucose and brain heart extract as feed, combined with high-pressure homogenization or ultrasonic disruption, a high-titer inactivated vaccine was prepared. The vaccine was quantified using nucleic acid quantification or P46 protein quantification. The vaccine efficacy was evaluated by the reduction in viral load in the tracheal mucosa of the immunized group and the challenge control group.
It improves the culture titer and immunization efficacy of inactivated Mycoplasma hyopneumoniae vaccine, avoids the safety issues of traditional inactivation methods, and makes vaccine efficacy evaluation more scientific. It is suitable for the preparation of single and multiple vaccines.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of veterinary biological products technology, and in particular to a method for preparing and evaluating the efficacy of an inactivated porcine mycoplasma pneumoniae vaccine. Background Technology
[0002] Mycoplasma pneumonia of swine (MPS), also known as swine enzootic pneumonia, is a highly contagious chronic respiratory infectious disease caused by Mycoplasma hyopneumoniae (Mhp). MPS is one of the most common and difficult-to-eradicate diseases in swine herds, causing slow growth, reduced growth rate, decreased feed conversion rate, and wasted feed and labor in infected pigs. Secondary infections can lead to severe mortality and significant economic losses. In large-scale pig farms, MPS often synergizes with several bacteria and viruses to cause swine respiratory disease syndrome, severely hindering the development of the pig industry. Furthermore, Mycoplasma hyopneumoniae can cause immunosuppression, leading to the failure of other vaccines. Pigs at different stages can be infected with mycoplasma pneumonia; it is highly contagious and has a high infection rate. MPS has long been considered one of the major infectious diseases threatening the global pig industry.
[0003] Currently, the main measures for controlling Mycoplasma pneumoniae infection in pigs are the use of antibiotics and vaccines. Clinical practice has shown that mycoplasma readily develops drug resistance, and antibiotic treatment cannot completely eradicate mycoplasma infection in pig herds. Therefore, vaccination is a more effective measure for controlling this disease.
[0004] Clinically used porcine mycoplasma pneumoniae vaccines are mainly divided into two types: inactivated vaccines and live attenuated vaccines. Several domestic and international companies produce porcine mycoplasma pneumoniae vaccines. However, the in vitro culture conditions for Mhp are demanding, requiring specific nutrients to maintain its growth and reproduction. Therefore, the in vitro culture of Mhp remains the primary challenge to be overcome in its vaccine preparation.
[0005] The lung lesion scoring method is commonly used to evaluate the efficacy of Mhp vaccines. However, this method is greatly affected by differences in experimental pig breeds, individual differences, other pathogens with similar lesion characteristics, and the subjective judgment of the evaluator. It is difficult to apply in practice and affects the scientific nature of vaccine efficacy evaluation. Summary of the Invention
[0006] The purpose of this invention is to provide a method for preparing and evaluating the efficacy of an inactivated porcine mycoplasma pneumoniae vaccine, in order to solve the problems existing in the prior art. By optimizing the fermentation process and treatment conditions for culturing porcine mycoplasma pneumoniae, the prepared inactivated porcine mycoplasma pneumoniae vaccine has the characteristics of high culture titer, excellent immunogenicity, and high safety. Furthermore, the protective effect of the inactivated porcine mycoplasma pneumoniae vaccine can be accurately evaluated by detecting the reduction in viral load in the tissues of immunized pigs.
[0007] To achieve the above objectives, the present invention provides the following solution:
[0008] This invention provides a method for preparing an inactivated porcine mycoplasma pneumoniae vaccine, comprising the following steps:
[0009] A strain of Mycoplasma hyopneumoniae was revived, and an antigenic bacterial solution was obtained through passage culture and fermentation culture. The antigenic bacterial solution was purified and quantified. Based on the quantification results, the bacterial solution was diluted, and then an adjuvant was added to prepare an inactivated Mycoplasma hyopneumoniae vaccine.
[0010] The fermentation process involves two feedings, which include the following components: coenzyme, PPLO, glucose, and brain and heart extract powder.
[0011] Preferably, the feed comprises the following components at the following concentrations by mass-volume percentage: 1-5% coenzyme, 5-10% PPLO, 5-10% glucose, 10-20% brain and heart extract powder, and water as the solvent.
[0012] More preferably, the coenzyme is 1-3%, PPLO is 8-10%, glucose is 8-10%, brain and heart extract is 10-12%, and the solvent is water.
[0013] More preferably, the coenzyme is 3%, PPLO is 10%, glucose is 10%, brain and heart extract is 10%, and the solvent is water.
[0014] Preferably, the fermentation conditions are: constant temperature culture at 37℃, tank pressure of 0.04MPa, rotation speed of 100r / min, and pH value of 7.6-7.8;
[0015] And / or the conditions for feeding are: both feedings are done when the pH of the fermentation culture system drops to 6.7, and the amount added is 0.1-0.5% of the total fermentation culture volume;
[0016] And / or after the second feeding, continue culturing for 8–15 hours, and harvest the fermentation broth when the pH drops to 6.4, which is the antigen broth.
[0017] Preferably, the purification method is as follows: the bacterial solution is centrifuged at high speed or concentrated by membrane centrifugation to obtain bacterial sludge, and then resuspended in PBS to obtain a 10-50 times concentrated solution; wherein, the pore size of the concentrated membrane is 800-1000 kDa, the high-speed centrifugation speed is 12000-16000 rpm, and the centrifugation is performed continuously for 3-5 times.
[0018] The quantification method is either nucleic acid quantification or P46 protein quantification.
[0019] Preferably, after purification of the antigen bacterial solution, a cell disruption step is included before quantification. The cell disruption is performed by either ultrasonic disruption or homogenization, preferably homogenization. The ultrasonic disruption parameters are 50–75 Hz for 5–15 min; the homogenization pressure is 400–1500 Bar.
[0020] Preferably, after dilution, the Mycoplasma hyopneumoniae antigen content in the bacterial solution is 0.5 × 10⁻⁶. 7.0 ~1.5×10 7.0 Number of copies / header;
[0021] The volume ratio of the diluted bacterial solution to the adjuvant is 9:1. The adjuvant includes one or more of the following: oil-in-water adjuvant, polymer and water adjuvant, water-in-oil adjuvant, aluminum hydroxide adjuvant, vitamin E adjuvant, or other adjuvants that can be used in inactivated vaccines for livestock.
[0022] This invention also provides a method for evaluating the efficacy of an inactivated porcine mycoplasma pneumoniae vaccine, comprising the step of comparing the reduction in viral load in the tracheal mucosa at the same location and area between the immunization group and the challenge control group to evaluate the immunization effect of the inactivated porcine mycoplasma pneumoniae vaccine; wherein the immunization group is a treatment group immunized with the inactivated porcine mycoplasma pneumoniae vaccine prepared by the aforementioned preparation method, and the challenge control group is a treatment group that has not received any vaccine immunization.
[0023] The present invention also provides an inactivated porcine mycoplasma pneumoniae vaccine prepared using the aforementioned preparation method.
[0024] The present invention also provides the application of the porcine mycoplasma pneumoniae inactivated vaccine in the preparation of bivalent or multivalent inactivated vaccines.
[0025] This invention also provides a bivalent inactivated vaccine, comprising the aforementioned porcine mycoplasma pneumoniae inactivated vaccine and porcine circovirus type 2 antigen; the p46 protein content of the porcine mycoplasma pneumoniae antigen is 50-100 μg / dose, and the amount of porcine circovirus type 2 antigen added is 0.5 × 10⁻⁶. 8.0 ~1.5×10 8.0 TCID 50 / Toufen.
[0026] The present invention discloses the following technical effects:
[0027] This invention increases the Mhp fermentation titer to 10 by timely supplementing the nutrient solution twice during the cultivation of Mycoplasma hyopneumoniae. 10.0 A CCU / ml or higher, and an efficient culture process, are beneficial for improving the immune efficacy of inactivated Mycoplasma hyopneumoniae vaccines.
[0028] The present invention provides a method for purifying porcine Mycoplasma pneumoniae bacterial suspension without inactivation. After purification, the bacterial cells are homogenized under high pressure or ultrasonically broken up. This method is beneficial for exposing the antigenic epitopes of porcine Mycoplasma pneumoniae, thereby improving the vaccine's immunization effect, while avoiding the vaccine safety issues caused by traditional formaldehyde inactivation.
[0029] The method for quantifying the antigen of the inactivated Mycoplasma pneumoniae vaccine of this invention is a nucleic acid assay or a P46 protein assay. First, the bacterial solution is concentrated, purified and broken up by 10 to 50 times. Then, the concentrated bacterial solution is diluted according to the test results, and the purified antigen is added quantitatively. This method is beneficial to improving the safety and batch-to-batch stability of the vaccine.
[0030] This invention evaluates the immunization efficacy of an inactivated porcine mycoplasma pneumoniae vaccine by comparing the reduction in viral load in the tracheal mucosa of the same location and area between the immunized group and the challenge control group. This avoids the influence of individual differences, mixed infections with other pathogens, and subjective judgments, resulting in a more scientific efficacy evaluation.
[0031] This invention provides a highly purified semi-finished porcine mycoplasma pneumoniae inactivated vaccine. The adjuvant can be an oil-in-water adjuvant, a polymer and water adjuvant, an oil-in-water adjuvant, aluminum hydroxide adjuvant, vitamin E adjuvant, or combinations thereof, as well as other adjuvants suitable for livestock inactivated vaccines. It exhibits no adverse immune reactions, good absorption, and meets animal food safety and animal welfare requirements. Detailed Implementation
[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0033] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0034] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0035] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.
[0036] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0037] The porcine mycoplasma pneumoniae culture medium used in the embodiments of the present invention is a mycoplasma solid or liquid culture medium, which was prepared by the inventors according to the formula on pages 32-33 of Appendix III of the Pharmacopoeia of the People's Republic of China (2020 edition).
[0038] Example 1: Preparation of porcine mycoplasma pneumoniae inactivated vaccine antigen
[0039] 1. The method for culturing Mycoplasma hyopneumoniae in porcine culture according to the present invention
[0040] (1) Preparation of culture medium and feed for Mycoplasma hyopneumoniae in pigs
[0041] Preparation of mycoplasma culture medium feed: Weigh appropriate amounts of coenzyme I, PPLO medium, glucose, brain heart extract powder, and sodium hydroxide and dissolve them in water. On a weight-volume percentage (W:V) basis, make coenzyme 3%, PPLO 10%, glucose 10%, and brain heart extract powder 10%. Filter through a 0.22μm membrane to remove bacteria and use as feed.
[0042] (2) Culture of Mycoplasma hyopneumoniae in swine pulmonary strain
[0043] After reviving the inactivated porcine mycoplasma pneumoniae vaccine (Strain J), it was inoculated into mycoplasma liquid culture medium at a volume ratio of 1:10. The pH was adjusted to 7.6–7.8, and the culture was passaged at a constant temperature of 37°C. When the pH of the culture dropped to 6.7, the bacterial culture was harvested as the primary seed culture. The primary seed culture was then inoculated and passaged again at a ratio of 1:10 using the same method, and the bacterial culture was harvested as the secondary seed culture.
[0044] The secondary seed culture was inoculated into mycoplasma liquid culture medium at a volume ratio of 1:10 and fermented in a mechanically stirred fermenter. The pH was adjusted to 7.7, and the fermentation was carried out at a constant temperature of 37°C, a tank pressure of 0.04 MPa, and a stirring speed of 100 r / min. After 15 hours of cultivation, when the pH dropped to 6.7, the first feeding was added, with an amount of 0.5% of the total culture volume, and the pH was adjusted to 7.7. After another 15 hours of cultivation, when the pH dropped to 6.7, feeding was added again, with an amount of 0.5% of the total culture volume. After another 15 hours of cultivation, the fermentation broth was harvested when the pH dropped to 6.4.
[0045] 2. Culture of Mycoplasma hyopneumoniae using conventional methods
[0046] The above secondary seed culture was inoculated into mycoplasma liquid culture medium at a volume ratio of 1:10. Fermentation was carried out in a mechanically stirred fermenter, with the pH adjusted to 7.7, and the culture was kept at a constant temperature of 37°C, a tank pressure of 0.04 MPa, and a rotation speed of 100 r / min. After 8–15 h of culture, the fermentation broth was harvested when the pH dropped to 6.4.
[0047] 3. Antigen content determination
[0048] As shown in Table 1 below, the *Mycoplasma hyopneumoniae* cultured using the method of this invention (see "1. Method for culturing *Mycoplasma hyopneumoniae* culture of this invention") showed higher CCU, nucleic acid content, and P46 protein content than those cultured using conventional methods (see "2. Conventional method for culturing *Mycoplasma hyopneumoniae* culture"). CCU was determined according to the method described in the Veterinary Pharmacopoeia, and nucleic acid and P46 protein content were detected using conventional commercial reagent kits.
[0049] Table 1. Results of antigen content determination using different culture methods
[0050]
[0051] Example 2: Preparation and efficacy testing of inactivated porcine mycoplasma pneumoniae vaccine
[0052] 1. Preparation of Mycoplasma hyopneumoniae antigen
[0053] The porcine mycoplasma pneumoniae strain J was produced by Ruipu (Baoding) Biopharmaceutical Co., Ltd., with an antigen titer of 10 before inactivation. 8.5 CCU. The preparation method is the same as in Example 1.
[0054] 2. Preparation of inactivated porcine mycoplasma pneumoniae vaccine
[0055] The *Mycoplasma hyopneumoniae* fermentation broth was centrifuged three times at 15,000 rpm using a continuous flow centrifuge, and the bacterial sludge was scraped off. The broth was resuspended in PBS to prepare a 50-fold concentrated bacterial solution, which was then homogenized at a pressure of 1000 Bar. The antigen content was determined using a nucleic acid quantification method, and the antigen concentration was 2.5 × 10⁻⁶. 8.0 Copy number / mL. Dilute the concentrated bacterial solution to achieve a Mycoplasma hyopneumoniae antigen content of 1.0 × 10⁻⁶. 7.0 Number of copies / heads.
[0056] Take 90 parts by weight of diluted bacterial solution, add 10 parts of oil-in-water adjuvant (SDA15A adjuvant was used in this experiment, but it is not limited to this), and emulsify to prepare porcine mycoplasma pneumonia inactivated vaccine.
[0057] 3. Efficacy evaluation of inactivated porcine mycoplasma pneumoniae vaccine
[0058] Fifteen 7-day-old specific pathogen-free healthy piglets were selected. In group I, 5 piglets were immunized by intramuscular injection of the inactivated porcine mycoplasma pneumoniae vaccine of this invention in the neck, 2.0 mL / piglet; in group II, 5 piglets were immunized by intramuscular injection of the inactivated porcine mycoplasma pneumoniae vaccine of this invention in the neck (mycoplasma antigen was not treated), 2.0 mL / piglet; and the other 5 piglets were not immunized and served as controls.
[0059] Fourteen days later, the immunized groups I and II were immunized a second time using the same dose and route. Fourteen days after the second immunization, both immunized and control pigs were challenged with the virus via intratracheal injection. The virus used for challenge was the lung lavage fluid from pigs infected with porcine mycoplasma pneumoniae, containing 1.0 × 10⁻⁶ Mycoplasma pneumoniae. 7.0 Number of copies / heads.
[0060] Clinical symptoms of experimental pigs were observed and recorded. Lung lesion scores were determined by necropsy 28 days after challenge, and the viral load in the tracheal wall was measured. During necropsy, the trachea and lung tissue were completely removed. Two square tracheal wall samples (1cm x 1cm) were taken from the dorsal and ventral sides at the upper margin of the tracheal branches. These samples were placed in 3.0 mL of Mycoplasma hyopneumoniae culture medium and incubated overnight before the Mycoplasma hyopneumoniae content was measured. The challenge control group should have at least three pigs with Mycoplasma hyopneumoniae content in the tracheal wall exceeding 1.0 × 10⁻⁶. 8.0 For the immunized group, at least 4 pigs should have a Mycoplasma pneumoniae content in their tracheal wall tissue below 1.0 × 10⁻⁶. 7.0 Copy number.
[0061] The results showed that 10-14 days after the challenge, one pig in immunization group I, two pigs in immunization group II, and four pigs in the challenge control group developed symptoms such as dry cough or wheezing, which persisted until necropsy on day 28.
[0062] The Mycoplasma pneumoniae content in the tracheal wall of all 5 pigs in the challenge control group was higher than 1.0 × 10⁻⁶. 8.0The copy number of Mycoplasma pneumoniae in the tracheal wall of 10 immunized pigs was less than 1.0 × 10⁻⁶. 7.0 The copy number was not correlated with lung lesion scores and symptoms such as dry cough and wheezing.
[0063] The protection rate of the vaccine of this invention (immunization group I) against challenge was 86%, and the protection rate of immunization group II (antigen untreated) against challenge was 78%, both significantly higher than that of the challenge control group. This indicates that the inactivated porcine mycoplasma pneumoniae vaccine of this invention has a good protective effect against the circulating strain, as shown in Table 2.
[0064] Table 2. Efficacy Evaluation Results of Inactivated Porcine Mycoplasma Pneumoniae Vaccine
[0065]
[0066] Example 2: Efficacy testing of the circovirus bivalent inactivated vaccine
[0067] 1. Preparation of porcine circovirus type 2 antigen
[0068] The porcine circovirus type 2 antigen is strain ZJ / C, produced by Ruipu (Baoding) Biopharmaceutical Co., Ltd., with an antigen titer of 10 before inactivation. 8.0 TCID 50 .
[0069] The porcine circovirus type 2 subunit antigen is a baculovirus-expressed Cap protein antigen, provided by Hunan Zhong'an Biopharmaceutical Co., Ltd.
[0070] 2. Preparation of Mycoplasma hyopneumoniae antigen
[0071] The porcine mycoplasma pneumoniae strain J was produced by Ruipu (Baoding) Biopharmaceutical Co., Ltd., with an antigen titer of 10 before inactivation. 8.5 CCU, cultured using the same method as in Example 1.
[0072] In this experiment, the P46 protein quantification method was used to add antigen. The fermentation broth was centrifuged at high speed using a continuous flow centrifuge, and the bacterial sludge was scraped off and resuspended in 1 / 10 volume of PBS. Samples were taken, and the antigen content was detected using the P46 protein quantification method. The results showed that the content of the porcine mycoplasma pneumoniae antigen P46 protein was 500 μg / mL. The antigen in the bacterial broth was ultrasonically disrupted in this experiment, with the power set at 50% and the time set at 15 min.
[0073] 3. Preparation of the circovirus bivalent inactivated vaccine
[0074] Mix porcine mycoplasma pneumoniae antigen, inactivated porcine circovirus type 2 (PCV2) antigen, and PBS thoroughly to achieve a porcine mycoplasma pneumoniae antigen P46 protein content of 50 μg / dose and a porcine circovirus type 2 antigen addition amount of 10 μg / dose. 8.0 TCID 50 / dose. By weight, take 1 part of diluted bacterial solution, add 2 parts of nano-aluminum gel adjuvant, and emulsify to prepare a porcine circovirus type 2 and porcine mycoplasma pneumonia bivalent inactivated vaccine (bivalent inactivated vaccine A).
[0075] Porcine mycoplasma pneumoniae antigen, porcine circovirus type 2 subunit antigen, and PBS were mixed thoroughly to achieve a porcine mycoplasma pneumoniae antigen P46 protein content of 50 μg / dose and a porcine circovirus type 2 subunit antigen addition of 100 μg / dose. One part by weight of the diluted bacterial solution was taken, and two parts by weight of nano-aluminum gel adjuvant were added, and emulsified to prepare a bivalent inactivated vaccine of porcine circovirus type 2 subunit and porcine mycoplasma pneumoniae (bivalent inactivated vaccine B).
[0076] 4. Evaluation of the efficacy of the bivalent inactivated vaccine
[0077] Fifteen 7-day-old specific pathogen-free piglets were divided into three groups: Group A (5 piglets) received intramuscular injection of the bivalent inactivated vaccine A of this invention in the neck (2.0 mL / piglet); Group B (5 piglets) received intramuscular injection of the bivalent inactivated vaccine B of this invention in the neck; and Group C (5 piglets) served as a challenge control without immunization.
[0078] Fourteen days later, the immunized group received a second immunization with the same dose and route.
[0079] Fourteen days after the second immunization, immunized pigs and control pigs were challenged with the prevalent porcine mycoplasma pneumoniae virus via intratracheal injection. The porcine mycoplasma pneumoniae content was 1.0 × 10⁻⁶. 7.0 Copy number / dosage; Immunized pigs and control pigs were challenged with the circulating porcine circovirus type 2 strain via intramuscular injection in the neck. The viral load of porcine circovirus type 2 was 10. 7.5 TCID 50 / Toufen.
[0080] Twenty-eight days after the viral challenge, an autopsy was performed to determine the effectiveness of the viral challenge protection.
[0081] Bivalent inactivated vaccines A and B both showed good protective effects against both PCV2 and Mhp, as shown in Table 3.
[0082] Table 3. Results of efficacy evaluation of the combined inactivated circovirus and trichomonal vaccine.
[0083]
[0084]
[0085] The vaccine preparation method of this invention is applicable to the preparation of inactivated Mycoplasma hyopneumoniae vaccines, and is not limited to the preparation of single inactivated Mycoplasma hyopneumoniae vaccines. It is also applicable to the preparation of bivalent or multivalent inactivated vaccines with other pathogenic microorganisms.
[0086] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. A method for preparing an inactivated porcine mycoplasma pneumoniae vaccine, characterized in that, Includes the following steps: A strain of Mycoplasma hyopneumoniae was revived, and an antigenic bacterial solution was obtained through passage culture and fermentation culture. The antigenic bacterial solution was purified and quantified. Based on the quantification results, the bacterial solution was diluted, and then an adjuvant was added to prepare an inactivated Mycoplasma hyopneumoniae vaccine. The fermentation process involves two feedings, which include the following components: coenzyme, PPLO, glucose, and brain and heart extract powder.
2. The preparation method according to claim 1, characterized in that, The feed comprises the following components at the following concentrations by weight-volume percentage: 1-5% coenzyme, 5-10% PPLO, 5-10% glucose, 10-20% brain and heart extract powder, and water as the solvent.
3. The preparation method according to claim 1, characterized in that, The fermentation conditions were as follows: constant temperature culture at 37℃, tank pressure of 0.04MPa, rotation speed of 100r / min, and pH value of 7.6-7.
8. And / or the conditions for feeding are: both feedings are done when the pH of the fermentation culture system drops to 6.7, and the amount added is 0.1-0.5% of the total fermentation culture volume; And / or after the second feeding, continue culturing for 8–15 hours, and harvest the fermentation broth when the pH drops to 6.4, which is the antigen broth.
4. The preparation method according to claim 1, characterized in that, The purification method is as follows: the bacterial solution is centrifuged at high speed or concentrated by membrane centrifugation to obtain bacterial sludge, and the concentrate is resuspended in PBS; wherein, the pore size of the concentrating membrane is 800-1000 kDa, the high-speed centrifugation speed is 12000-16000 rpm, and the centrifugation is repeated 3-5 times. The quantification method is either nucleic acid quantification or P46 protein quantification.
5. The preparation method according to claim 1, characterized in that, After purification of the antigen bacterial solution, a cell disruption step is included before quantification.
6. The preparation method according to claim 1, characterized in that, After dilution, the Mycoplasma hyopneumoniae antigen content in the bacterial suspension was 0.5 × 10⁻⁶. 7.0 ~1.5×10 7.0 Number of copies / header; And / or the volume ratio of diluted bacterial solution to adjuvant is 9:
1.
7. A method for evaluating the efficacy of an inactivated porcine mycoplasma pneumoniae vaccine, characterized in that, The method includes steps to evaluate the immunization effect of the inactivated porcine mycoplasma pneumoniae vaccine by comparing the reduction in viral load in the tracheal mucosa at the same location and area between the immunization group and the challenge control group; wherein the immunization group is a treatment group immunized with the inactivated porcine mycoplasma pneumoniae vaccine prepared by any one of claims 1-6, and the challenge control group is a treatment group that has not received any vaccine immunization.
8. An inactivated porcine mycoplasma pneumoniae vaccine prepared by the preparation method according to any one of claims 1-6.
9. The use of the porcine mycoplasma pneumoniae inactivated vaccine of claim 8 in the preparation of a bivalent or multivalent inactivated vaccine.
10. A bivalent inactivated vaccine, characterized in that, The vaccine includes the inactivated porcine mycoplasma pneumoniae vaccine as described in claim 8 and porcine circovirus type 2 antigen; the P46 protein content of the porcine mycoplasma pneumoniae antigen is 50-100 μg / dose, and the amount of porcine circovirus type 2 antigen added is 0.5 × 10⁻⁶. 8.0 ~1.5×10 8.0 TCID 50 / Toufen.